There are a number of amphiphilic probes available for the staining of cellular membranes which cover the UV and visible region of the spectrum. However, the commonly used membrane interactive fluorophores have fluorescent signatures which overlap those of other fluorophores used as molecular tags for examining aspects of cell biology or biological structures, including biomolecules such as proteins or nucleic acids. Due to this overlap in spectral ranges it is not possible to selectively visualize stained membrane structures without exciting at the same time other labelled cell structures or biomolecules and vice versa. However, the possibility of selective excitation of different fluorophores having non-overlapping or only marginally overlapping absorption/emission ranges is a prerequisite for the study of transport processes between different cell compartments. Moreover, the staining of membranes in plant cells using fluorophores having their absorption/emission ranges in the UV or visible region of the spectrum is problematic, since the background emission caused by naturally occurring fluorophores, such as the autofluorescence of chlorophyll, drastically reduces the signal/noise ratio which leads to a highly decreased sensitivity of the method.
Particularly, those fluorophores usually employed in the staining of membranes, such as DII, DIO or DIA have the disadvantage that their spectral ranges overlap the spectral ranges of the most common fluorescence markers such as green fluorescent protein (GFP) or YFP which have both for example emission ranges of 530 nm and below.
Selected derivatives of anthraquinone have been reported as fluorophores which display a long-wavelength emission extending into the infrared region of the spectrum. WO 99/65992 describes for example anthraquinone fluorophores which are able to stain the nucleus of a cell through intercalating into nucleic acids present in the cell nucleus. However, the anthraquinone derivatives described in WO 99/65992 are not suited to stain membranes of living or fixed cells and, moreover, the production of the respective anthraquinones is only possible in low overall yields.
Therefore, a constant need exists for a membrane interactive fluorophore having an absorption/emission range which does not or substantially not overlap with the spectral ranges of the most commonly used fluorophores which are activated in the UV or visible region.